This proposal constitutes a renewal application to study the role of the HIV-1 Vpr gene product. The overall goal of the studies proposed here is to understand the interaction between the various functions of Vpr and how they contribute to the pathogenesis of HIV-1. The hypothesis to be addressed here is that Vpr plays a critical role for HIV-1 replication and pathogenesis. The HIV-1 Vpr gene product is a 96 amino acid protein, which is important for viral replication and likely to be critical for HIV-1 pathogenesis. Vpr is expressed following infection of cells and is also found packaged in virions. In 1995, at the beginning of the initial funding period of this grant, we were among the first to describe a novel phenotype of the Vpr protein whereby cells infected by HIV-1 expressing Vpr undergo arrest at the G2 phase of the cell cycle. We focused our efforts on further characterizing this unique function of the Vpr protein. We have shown that cell cycle arrest is a highly evolutionarily conserved property of Vpr. In addition, we found that cell cycle arrest induced by Vpr is followed by apoptosis of the arrested cells. Other investigators have reported that Vpr enhances virus production and is involved in cytokine regulation. During the past funding period we made the important observation that Vpr that is packaged into virions is capable of inducing both cell cycle arrest and apoptosis independent of subsequent events in the viral lifecycle. Recently, we made the observation that virion Vpr is critical for expression from unintegrated viral DNA. Thus, in combination these results indicate that Vpr plays an important role in HIV-1 pathogenesis through the immediate early upregulation of viral gene expression and longer term effects upon T-cell metabolism. During the previous funding periods we have generated a considerable amount of preliminary data about Vpr action, developed a number of unique assays for assessment of Vpr function, and developed reagents which will allow us to effectively carry forward further studies on the mechanism of Vpr action. The specific aims are: Aim 1) To further characterize cell cycle arrest and apoptosis mediated by Vpr. Aim 2) To characterize the newly described process of Vpr mediated transactivation of unintegrated HIV-1 DNA. Aim 3) To further characterize the role of Vpr in viral pathogenesis.